PROJECT SUMMARY/ABSTRACT Relapsing fever (RF) is a globally-important, neglected bacterial zoonosis that is commonly misdiagnosed and underreported. RF, named for the hallmark symptom of repeated rounds of febrile illness that the patients experience as a result of recurring episodes of bacteremia, develops after spirochetes of the genus Borrelia are transmitted to humans via the bite of a colonized human body louse or tick (tick-borne RF, TBRF). RF spirochetes are divided into Old World and New World groups based on the parts of the world where they are endemic, and this division is further supported by phylogenetic comparisons. In addition to the geographic and genotypic separation, there are aspects of their pathogenesis and vector-pathogen interactions that are unique between the two groups of RF spirochetes. Borrelia crocidurae and Borrelia duttonii represent two prominent Old World TBRF spirochetes that are common to Africa, while Borrelia hermsii and Borrelia turicatae from the Americas are the major New World TBRF spirochetes. Although the etiological agents of RF were identified over 100 years ago, information is limited regarding the molecular mechanisms contributing to their pathogenesis and enzootic lifecycle. Old World TBRF Borrelia account for the majority of disease worldwide, but most RF research to date has focused on New World TBRF spirochetes. As such, there is an obvious need to expand our study of RF spirochete biology to include the more relevant Old World TBRF Borrelia. Koch's molecular postulates are a cornerstone of contemporary molecular microbiology, and the application of molecular techniques and genetic manipulation represents one of the most direct approaches to identify and characterize bacterial virulence determinants. Once bacterial factors required during the RF spirochete enzootic cycle have been identified, we can begin to study their physiological contributions to these bacterial processes and identify viable targets against which diagnostics or therapies could be developed. To date, methods of genetic manipulation to study RF Borrelia have only been applied to the New World TBRF spirochetes, B. hermsii and B. turicatae. As such, our limited knowledge regarding the pathogenic mechanisms and vector interactions involved in the enzootic cycle of Old World TBRF Borrelia is largely due to the lack of the requisite systems for genetic manipulation of these other RF species. To address this deficiency, we propose two Specific Aims. In Specific Aim 1, we will use standard recombinant DNA and cloning techniques to develop tools for genetic manipulation of the Old World TBRF spirochete, B. duttonii. These tools include selectable resistance markers, shuttle vectors, and constructs for allelic exchange mutagenesis and integration. In Specific Aim 2, techniques for transformation of B. duttonii will be defined using methods developed for other Borrelia species. Once the molecular tools and genetic transformation techniques have been developed, we will be poised to study pathogenesis and vector-pathogen interactions in a relevant Old World TBRF spirochete.